ABSTRACT
OBJECTIVES: Our objective was to compare the interactions of red wine and grapefruit juice with cisapride. METHODS: The oral pharmacokinetics of cisapride, its norcisapride metabolite, and electrocardiographic QTc interval were determined over a 24-hour period after administration of cisapride 10 mg with 250 mL grapefruit juice, red wine (cabernet sauvignon), or water in a randomized 3-way crossover study in 12 healthy men. RESULTS: The cisapride area under the concentration-time curve (AUC) and the maximum plasma drug concentration after single-dose administration (C(max)) with grapefruit juice were 151% (P <.01) and 168% (P <.001), respectively, of those with water. The increase in cisapride AUC and C(max) was variable among individuals; however, cisapride AUC and C(max) were enhanced by the same proportion. The time to reach maximum concentration after drug administration (t(max)) and the apparent elimination half-life (t((1/2)) for cisapride and the pharmacokinetics of norcisapride were not altered. Norcisapride/cisapride ratios were reduced. Cisapride AUC and C(max) with red wine were 115% (difference not statistically significant) and 107% (difference not statistically significant), respectively, of those with water. The cisapride t(max) was slightly longer. Cisapride t((1/2)) and norcisapride pharmacokinetics were not different. The norcisapride/cisapride ratio at cisapride C(max) was lower. One subject had a doubling in cisapride AUC and C(max) and a decrease in norcisapride/cisapride ratios with red wine and also had the largest interaction with grapefruit juice. QTc interval was unchanged in all treatment groups and individuals. CONCLUSIONS: A single glass of grapefruit juice produced an individual-dependent variable increase in the systemic availability of cisapride by inhibition of intestinal cytochrome P450 3A4 (CYP3A4) activity. The identical volume of red wine caused only minor changes in cisapride pharmacokinetics despite some inhibition of CYP3A4 in most individuals. However, even this amount of red wine may cause a marked interaction similar to that for grapefruit juice in individuals with a preexisting high intestinal CYP3A4 content.
Subject(s)
Beverages , Cisapride/analogs & derivatives , Cisapride/pharmacokinetics , Citrus , Cytochrome P-450 Enzyme Inhibitors , Gastrointestinal Agents/pharmacokinetics , Mixed Function Oxygenases/antagonists & inhibitors , Wine , Adult , Area Under Curve , Cisapride/blood , Cisapride/pharmacology , Cross-Over Studies , Cytochrome P-450 CYP3A , Food-Drug Interactions , Gastrointestinal Agents/blood , Gastrointestinal Agents/pharmacology , Humans , Male , Reference ValuesABSTRACT
We have previously demonstrated depressed vascular contractility in intralobar pulmonary artery (PA) rings isolated from rats with acute Pseudomonas pneumonia. Here we describe the role of arachidonic acid (AA) metabolites in the regulation of pulmonary vascular tone in inflammation. Pneumonia was induced by intratracheal injection of P. aeruginosa organisms. Rats were sacrificed 44 h later. EETs and 20-HETE were formed at significantly lower rates in pneumonia compared with control lung microsomes. Vasoactive effects of CYP metabolites (5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, and 20-HETE) on small PA rings from control or pneumonia rats were assessed in vitro. All four EETs and 20-HETE were more potent PA vasoconstrictors than KCl or phenylephrine (PE). However, this potency was attenuated in PA rings from pneumonia lungs compared with control. In contrast, pneumonia had no effect on COX activity [total pulmonary prostaglandin (PG), PGE(2), and 6-keto-PGF(1 alpha)]. In vitro vascular contractility to KCl, PE, or PGF(2 alpha) was assessed in small PA rings from control and pneumonia rats in the presence and absence of the COX-2 inhibitor NS-398 (10 microM). NS-398 did not reverse the attenuated contractile responses to KCl, PE, or PGF(2 alpha) in pneumonia rats. Nitrite/nitrate levels, inducible nitric-oxide synthase and heme oxygenase activities were all significantly elevated in pneumonia lungs. In conclusion, vasodilator PGs produced by COX-2 do not contribute to the depressed PA contractility in this model of pneumonia. Depressed pulmonary production and vasoconstrictor effects of CYP metabolites of AA (possibly due to increased NO and/or carbon monoxide) indicate a potential role for these vasoactive metabolites in this model of acute pneumonia.
Subject(s)
Arachidonic Acid/metabolism , Cytochrome P-450 Enzyme System/metabolism , Isoenzymes/metabolism , Pneumonia, Bacterial/physiopathology , Prostaglandin-Endoperoxide Synthases/metabolism , Pseudomonas Infections/physiopathology , Pulmonary Circulation/physiology , Animals , Cyclooxygenase 2 , Heme Oxygenase (Decyclizing)/metabolism , Hydroxyeicosatetraenoic Acids/pharmacology , Lung/drug effects , Lung/metabolism , Male , Microsomes/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Nitrobenzenes/pharmacology , Pneumonia, Bacterial/metabolism , Prostaglandin Antagonists/pharmacology , Pseudomonas Infections/metabolism , Rats , Rats, Sprague-Dawley , Sulfonamides/pharmacologyABSTRACT
OBJECTIVES: To determine whether unprocessed grapefruit can cause a drug interaction, whether the active ingredients are naturally occurring, and whether specific furanocoumarins or flavonoids are involved. METHODS: The oral pharmacokinetics of felodipine and its dehydrofelodipine metabolite were determined after administration of felodipine 10 mg extended-release tablet with 250 mL commercial grapefruit juice, homogenized grapefruit segments, or extract of segment-free parts equivalent to one unprocessed fruit or water in a randomized four-way crossover study. Inhibition of recombinant CYP3A4 by furanocoumarins (bergamottin, 6',7'-epoxybergamottin, 6',7'-dihydroxybergamottin) and flavonoids (naringenin optical isomers) was determined. Furanocoumarin and naringenin precursor (naringin) concentrations were measured in each grapefruit treatment. RESULTS: Felodipine AUC with commercial grapefruit juice, grapefruit segments, or grapefruit extract was on average 3-fold higher than that with water. Felodipine peak concentration was higher, but the half-life was unchanged. The dehydrofelodipine/felodipine AUC ratio was reduced. The furanocoumarins produced mechanism-based and competitive inhibition of CYP3A4. Bergamottin was the most potent mechanism-based inhibitor. Naringenin isomers produced only competitive inhibition. Bergamottin, 6',7'-dihydroxybergamottin, and naringin concentrations varied among grapefruit treatments but were sufficient to inhibit markedly in vitro CYP3A4 activity. CONCLUSIONS: Unprocessed grapefruit can cause a drug interaction with felodipine. The active ingredients are naturally occurring in the grapefruit. Bergamottin is likely important in drug interactions with commercial grapefruit juice. 6',7'-Dihydroxybergamottin and naringin may be more important in grapefruit segments because they are present in higher concentrations. Any therapeutic concern for a drug interaction with commercial grapefruit juice should now be extended to include whole fruit and possibly confectioneries made from grapefruit peel.
Subject(s)
Citrus , Felodipine/pharmacokinetics , Flavanones , Administration, Oral , Adult , Antioxidants/pharmacology , Area Under Curve , Beverages , Cross-Over Studies , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme Inhibitors , Delayed-Action Preparations , Drug Interactions , Felodipine/analogs & derivatives , Female , Flavonoids/pharmacology , Furocoumarins/pharmacology , Half-Life , Humans , Male , Mixed Function Oxygenases/antagonists & inhibitors , Radiation-Sensitizing Agents/pharmacologyABSTRACT
Renal microsomal cytochrome P-450 monooxygenase-dependent metabolism of arachidonic acid generates a series of regioisomeric epoxyeicosatrienoic acids that can be further metabolized by soluble epoxide hydrolase to the corresponding dihydroxyeicosatrienoic acids. Evidence exists that these metabolites affect renal function and, in particular, blood pressure regulation. To examine this possibility, blood pressure and renal arachidonic acid metabolism were examined in mice with a targeted disruption of the soluble epoxide hydrolase gene. Systolic blood pressure of male soluble epoxide hydrolase-null mice was lower compared with wild-type mice in both the absence and presence of dietary salt loading. Both female soluble epoxide hydrolase-null and wild-type female mice also had significantly lower systolic blood pressure than male wild-type mice. Renal formation of epoxyeicosatrienoic and dihydroxyeicosatrienoic acids was markedly lower for soluble epoxide hydrolase-null versus wild-type mice of both sexes. Although disruption of soluble epoxide hydrolase in female mice had minimal effects on blood pressure, deletion of this gene feminized male mice by lowering systolic blood pressure and altering arachidonic acid metabolism. These data provide the first direct evidence for a role for soluble epoxide hydrolase in blood pressure regulation and identify this enzyme as a novel and attractive target for therapeutic intervention in hypertension.
Subject(s)
Blood Pressure/physiology , Epoxide Hydrolases/physiology , Animals , Arachidonic Acid/metabolism , Epoxide Hydrolases/genetics , Epoxide Hydrolases/metabolism , Hypertension/enzymology , Hypertension/physiopathology , Male , Mice , Mice, Knockout , SolubilityABSTRACT
In this investigation, we tested the hypothesis that the cytochrome P-450 (CYP) inhibitor 1-aminobenzotriazole (ABT) alters the susceptibility of rats to hyperoxic lung injury. Male Sprague-Dawley rats were treated i.p. with ABT (66 mg/kg), i.v. with N-benzyl-1-aminobenzotriazole (1 micromol/kg), or the respective vehicles, followed by exposure to >95% oxygen for 24, 48, or 60 h. Pleural effusion volumes were measured as estimates of hyperoxic lung injury, and lung microsomal ethoxyresorufin O-deethylation (EROD) (CYP1A1) activities and CYP1A1 apoprotein levels were determined by Western blotting. ABT-pretreated animals exposed to hyperoxia died between 48 and 60 h, whereas no deaths were observed with up to 60 h of hyperoxia in vehicle-treated animals. In addition, three of four ABT-treated rats exposed to hyperoxia for 48 h showed marked pleural effusions. Exposure of vehicle-treated rats to hyperoxia led to 6.3-fold greater lung EROD activities and greater CYP1A1 apoprotein levels than in air-breathing controls after 48 h, but both declined to control levels by 60 h. Liver CYP1A1/1A2 enzymes displayed responses to hyperoxia and ABT similar to the effects on lung CYP1A1. N-Benzyl-1-aminobenzotriazole markedly inhibited lung microsomal pentoxyresorufin O-depentylation (principally CYP2B1) activities in air-breathing and hyperoxic animals but did not affect lung EROD or liver CYP activities. In conclusion, the results suggest that induction of CYP1A enzymes may serve as an adaptive response to hyperoxia, and that CYP2B1, the major pulmonary CYP isoform, does not contribute significantly to hyperoxic lung injury.
Subject(s)
Cytochrome P-450 Enzyme Inhibitors , Hyperoxia/pathology , Oxygen/toxicity , Respiratory Distress Syndrome/chemically induced , Triazoles/pharmacology , Animals , Blotting, Western , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP1A2/metabolism , Drug Synergism , Enzyme Inhibitors/pharmacology , Hyperoxia/mortality , Liver/drug effects , Liver/enzymology , Male , Microsomes/drug effects , Microsomes/metabolism , Oxazines/pharmacokinetics , Pleural Effusion , Protein Isoforms/pharmacology , Rats , Rats, Sprague-Dawley , Respiratory Distress Syndrome/mortality , Time FactorsABSTRACT
Male Sprague-Dawley rats were treated intraperitoneally with corn oil, the aryl hydrocarbon receptor (AHR) agonist beta-naphthoflavone (betaNF), or the relatively weak AHR agonist alpha-naphthoflavone (alphaNF). Animals treated with betaNF experienced a significant loss (12%) of total body mass over 5 days and a dramatic elevation of CYP1A1 mRNA in all of the organs studied. Treatment with alphaNF had no significant effect on body mass after 5 days and caused only minor increases of liver, kidney, and heart CYP1A1 mRNA. In contrast, lung CYP1A1 mRNA was increased by alphaNF treatment to levels comparable to that seen with betaNF treatment. CYP2E1 mRNA levels were also elevated in liver, lung, kidney, and heart in response to betaNF treatment, whereas alphaNF was without effect. Large increases of CYP1Al-dependent 7-ethoxyresorufin O-deethylation (EROD) activity occurred with microsomes prepared from the tissues of betaNF-treated animals. Comparatively small changes were associated with alphaNF treatment, with the exception of lung, where EROD activity was increased to approximately 60% of that with betaNF treatment. CYP2E1-dependent p-nitrophenol hydroxylase (PNP) activity was also increased by betaNF treatment in microsomes prepared from kidney (3.1-fold), whereas alphaNF was without effect. In contrast, alphaNF or betaNF treatment caused significant decreases of lung microsomal PNP (72% and 27% of corn oil control, respectively) and 7-pentoxyresorufin O-deethylation (48% and 17% of corn oil control, respectively) activities, indicating that PNP activity may be catalyzed by P450 isoforms other than CYP2E1 in rat lung. We conclude that betaNF and alphaNF have differential effects on the expression and catalytic activity of CYP1A1 and CYP2E1, depending upon the organ studied. These changes most likely occur as a result of the direct actions of these compounds as AHR agonists, in addition to secondary effects associated with AHR-mediated toxicity.
Subject(s)
Benzoflavones/pharmacology , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP2E1/genetics , Gene Expression Regulation, Enzymologic/drug effects , Kidney/enzymology , Liver/enzymology , Lung/enzymology , Microsomes/enzymology , Myocardium/enzymology , Transcription, Genetic/drug effects , beta-Naphthoflavone/pharmacology , Animals , Body Weight/drug effects , Cytochrome P-450 CYP1A1/biosynthesis , Cytochrome P-450 CYP2E1/biosynthesis , Kidney/drug effects , Liver/drug effects , Lung/drug effects , Male , Microsomes, Liver/enzymology , Organ Size/drug effects , RNA, Messenger/genetics , Rats , Rats, Sprague-DawleyABSTRACT
OBJECTIVE: To test whether naringin or 6',7'-dihydroxybergamottin is a major active substance in grapefruit juice-felodipine interaction in humans. METHODS: Grapefruit juice was separated by means of centrifugation and filtration into supernatant and particulate fractions, which were then assayed for naringin and 6',7'-dihydroxybergamottin. The effect of these fractions, grapefruit juice (containing comparable amounts of both fractions), and water on the pharmacokinetics of oral felodipine were assessed in 12 healthy men in a randomized, 4-way crossover study. RESULTS: The amounts of naringin and 6',7'-dihydroxybergamottin in the supernatant fraction (148 mg and 1.85 mg) were greater than in the particulate fraction (7 mg and 0.60 mg). The area under the plasma concentration-time curve (AUC) and the peak concentration (Cmax) of felodipine were higher with supernatant fraction (81 nmol.h/L and 20 nmol/L), particulate fraction (117 nmol.h/L and 24 nmol/L), and grapefruit juice (130 nmol.h/L and 33 nmol/L) compared with water (53 nmol.h/L and 11 nmol/L). However, the supernatant fraction had a lower AUC for felodipine and a similar Cmax of felodipine relative to the particulate fraction. The supernatant fraction neither augmented the AUC of the primary metabolite dehydrofelodipine nor decreased the AUC ratio of dehydrofelodipine to felodipine compared with water. Individually the supernatant fraction consistently produced lower felodipine AUC and Cmax compared with grapefruit juice. In contrast, the particulate fraction had values ranging from more than grapefruit juice to less than supernatant fraction. CONCLUSIONS: Naringin and 6',7'-dihydroxybergamottin are not the major active ingredients, although they may contribute to the grapefruit juice-felodipine interaction. The variable effect with the particulate fraction may result from erratic bioavailability of unidentified primary active substances. The findings show the importance of in vivo testing to determine the ingredients in grapefruit juice responsible for inhibition of cytochrome P450 3A4 in humans.
Subject(s)
Anti-Arrhythmia Agents/pharmacokinetics , Antioxidants/pharmacology , Calcium Channel Blockers/pharmacokinetics , Citrus/metabolism , Cytochrome P-450 Enzyme Inhibitors , Enzyme Inhibitors/pharmacology , Felodipine/pharmacokinetics , Flavanones , Flavonoids/pharmacology , Furocoumarins/pharmacology , Mixed Function Oxygenases/antagonists & inhibitors , Administration, Oral , Adult , Anti-Arrhythmia Agents/blood , Area Under Curve , Beverages , Biological Availability , Calcium Channel Blockers/blood , Cross-Over Studies , Cytochrome P-450 CYP3A , Felodipine/blood , Food-Drug Interactions , Humans , Male , Reference ValuesABSTRACT
N-Aralkylated derivatives of 1-aminobenzotriazole are well-established, mechanism-based inhibitors of cytochrome P450 (CYP or P450). In this study, the kinetics of inactivation of CYP2B-dependent 7-pentoxyresorufin O-depentylation (PROD) and CYP1A-dependent 7-ethoxyresorufin O-deethylation (EROD) activities by enantiomers of N-(alpha-methylbenzyl)-1-aminobenzotriazole (alphaMB) were compared. The racemic mixture (+/-)-alphaMB, as well as the enantiomers (-)-alphaMB and (+)-alphaMB, produced a time-, concentration-, and NADPH-dependent loss of PROD and EROD activity in hepatic microsomes from phenobarbital-treated guinea pigs. The rates of PROD inactivation by (-)-alphaMB were significantly faster than for (+)-alphaMB. Consistent with this, the derived maximal kinact was also significantly greater for (-)-alphaMB than for (+)-alphaMB (0.49 vs. 0.35 min-1). In contrast, the concentrations required for the half-maximal rate of inactivation (Ki) were equivalent for (-)-alphaMB and (+)-alphaMB, whereas the degree of competitive inhibition of PROD activity was greater for (+)-alphaMB. No significant differences were found among (-)-alphaMB, (+)-alphaMB, and (+/-)-alphaMB with respect to mechanism-based inactivation (kinact = 0.18, 0.16, and 0.17 min-1, respectively) or competitive inhibition of EROD activity. No differences were found for the maximal extent of PROD or EROD inhibition or the loss of spectral P450 after an extended 30-min incubation with the inhibitors. We conclude that mechanism-based inactivation of guinea pig CYP2B, but not CYP1A, isozymes by alphaMB occurs in a stereoselective manner, most likely as a result of a difference in the balance between metabolic activation and deactivation for the alphaMB enantiomers.
Subject(s)
Cytochrome P-450 CYP1A1/antagonists & inhibitors , Cytochrome P-450 CYP2B1/antagonists & inhibitors , Microsomes, Liver/enzymology , Triazoles/pharmacology , Animals , Guinea Pigs , Half-Life , Isoenzymes/antagonists & inhibitors , Stereoisomerism , Triazoles/chemistryABSTRACT
Heme metabolism normally involves enzymatic conversion to biliverdin and subsequently to bilirubin, catalyzed by heme oxygenase and biliverdin reductase, respectively. We examined the ability of exogenously added hemin, biliverdin, or bilirubin to regulate Cyp1a1, an enzyme that may be active in bilirubin elimination. A substantial dose-dependent increase in Cyp1a1 mRNA occurred after treatment of Hepa 1c1c7 cells with either of the three compounds. This increase was readily apparent 1 hr after treatment with biliverdin or bilirubin but required >/=2 hr with hemin. Treatment of Hepa 1c1c7 cells with these compounds also caused a dose-dependent increase in Cyp1a1-dependent 7-ethoxyresorufin-O-deethylase (EROD) activity. Of the three compounds, bilirubin produced the greatest maximal increase in Cyp1a1 mRNA and EROD (5.5-, 10.5-, and 15-fold for 100 microM hemin, biliverdin, and bilirubin, respectively) activity. The RNA polymerase inhibitor actinomycin D completely blocked Cyp1a1 induction by these compounds, indicating a requirement for de novo RNA synthesis via transcriptional activation. The protein synthesis inhibitor cycloheximide did not affect Cyp1a1 mRNA induction, indicating a lack of requirement for labile protein factors. In contrast, EROD induction by hemin, biliverdin, or bilirubin was completely blocked by cycloheximide treatment, indicating that the increase in enzyme activity is dependent on increased Cyp1a1 apoprotein synthesis. Aryl hydrocarbon receptor (AHR)- and AHR nuclear translocator-deficient mutant Hepa 1c1c7 cells did not exhibit increased Cyp1a1 mRNA or EROD activity after treatment with these compounds, indicating the requirement for a functional AHR for this response. Consistent with this, hemin, biliverdin, and bilirubin were able to induce expression of the dioxin-response element/luciferase reporter plasmid pGudLuc1.1 after transient transfection into wild-type Hepa 1c1c7 cells. Gel retardation assays demonstrated that bilirubin, but not hemin or biliverdin, was able to transform the AHR to a form capable of specifically binding to a 32P-labeled oligonucleotide containing a dioxin-response element sequence. These data indicate that bilirubin induces Cyp1a1 gene transcription through direct interaction with the AHR. In contrast, hemin and biliverdin seem to induce Cyp1a1 indirectly by serving as precursors to the endogenous formation of bilirubin via normal heme metabolism pathways. This is the first direct demonstration that the endogenous heme metabolite bilirubin can directly regulate Cyp1a1 gene expression and enzymatic activity in an AHR-dependent manner.
Subject(s)
Bilirubin/pharmacology , Cytochrome P-450 CYP1A1/biosynthesis , DNA-Binding Proteins , Gene Expression Regulation, Enzymologic/drug effects , Liver Neoplasms, Experimental/enzymology , Oxidoreductases Acting on CH-CH Group Donors , Receptors, Aryl Hydrocarbon/metabolism , Animals , Aryl Hydrocarbon Receptor Nuclear Translocator , Biliverdine/pharmacology , Cytochrome P-450 CYP1A1/genetics , Enzyme Induction , Genes, Reporter , Heme/metabolism , Hemin/pharmacology , Liver Neoplasms, Experimental/pathology , Mice , Oxidoreductases/metabolism , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/genetics , Transcription Factors/genetics , Tumor Cells, CulturedABSTRACT
The effect of mutating Gly 478 to Ala in rat cytochrome P450 2B1 on the metabolism of N-benzyl-1-aminobenzotriazole was investigated. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of the wild-type enzyme was completely inactivated by incubating with 1 microM BBT. The G478A mutant, however, was not inactivated by incubating with up to 10 microM BBT. Whereas metabolism of BBT by the wild-type 2B1 resulted in the formation of benzaldehyde, benzotriazole, aminobenzotriazole, and a new metabolite, the G478A mutant generated only the later. This metabolite was found by NMR, IR, and mass spectrometry to be a dimeric product formed from the reaction of two BBT molecules. Two spectral binding constants, a high-affinity constant that was the same for both enzymes (30-39 microM) and a low-affinity constant that was 5-fold lower for the mutant enzyme (0.3 mM vs 1.4 mM), were observed with BBT. The apparent Km and kcat values for the G478A mutant with BBT were 0.3 mM and 12 nmol (nmol of P450)-1 min-1, respectively. Molecular modeling studies of BBT bound in the active site of P450 2B1 suggested that a mutation of Gly 478 to Ala would result in steric hindrance and suppress oxidation of BBT at the 1-amino nitrogen. When BBT was oriented in the 2B1 active site such that oxidation at the 7-benzyl carbon could occur, no steric overlap between Ala 478 and the substrate was observed. Thus, this orientation of BBT would be preferred by the mutant leading to oxidation at the 7-benzyl carbon and subsequent dimer formation. These findings indicate that a glycine 478 to alanine substitution in P450 2B1 altered the binding of BBT such that inactivating BBT metabolites were no longer generated.
Subject(s)
Cytochrome P-450 CYP2B1/metabolism , Glycine/metabolism , Triazoles/metabolism , Animals , Binding Sites , Chromatography, High Pressure Liquid , Heme/chemistry , Magnetic Resonance Spectroscopy , Male , Mass Spectrometry , Microsomes, Liver/enzymology , Models, Chemical , Models, Molecular , Mutagenesis, Site-Directed , Rats , Rats, Inbred F344 , Rats, WistarABSTRACT
The metabolism and covalent binding of radioactivity to microsomal protein of the cytochrome P450 (P450) mechanism-based inhibitors 1-amino-[14C]-2,3-benzotriazole ([14C]ABT) and two radiolabeled forms of N-benzyl-1-aminobenzotriazole (BBT), N-benzyl-1-amino-[14C]-2,3-benzotriazole ([14C]-2,3-BBT) and [14C]-N-7-benzyl-1-aminobenzotriazole ([14C]-7-BBT), were examined in hepatic or pulmonary microsomes from untreated and phenobarbital (PB)- or beta-naphthoflavone (betaNF)-induced guinea pigs. [14C]-2,3-BBT and [14C]-7-BBT were converted to multiple metabolites including ABT, benzotriazole, benzaldehyde, 2- or 3-hydroxy-BBT, and 4-hydroxy-BBT by hepatic microsomes, while [14C]ABT, whose primary metabolite was benzotriazole, underwent little biotransformation. Neither ABT nor BBT was extensively metabolized by pulmonary microsomes. Hepatic microsomes from betaNF (vs PB)-treated guinea pigs metabolized [14C]ABT, [14C]-2,3-BBT, and [14C]-7-BBT more extensively. The degree of NADPH-dependent covalent binding of [14C]-2,3-BBT- or [14C]-7-BBT-derived radioactivity (1.0 nmol/mg of protein) was higher than that of [14C]ABT (0.3-0.8 nmol/mg of protein) in hepatic microsomes, especially those from PB-induced animals. Covalent binding per nmol of P450 in pulmonary microsomes was 3-4-fold higher with [14C]-2,3-BBT (2.9 nmol/nmol of P450) than with [14C]-7-BBT (1.0 nmol/nmol of P450), whereas in hepatic microsomes from PB- or betaNF-treated animals the ratio of binding with the two forms of BBT was approximately 1:1. [14C]-2,3-BBT- and [14C]-7-BBT-derived radioactivity was covalently bound to proteins that migrated in the molecular weight region corresponding to P450 on SDS-PAGE following incubation with NADPH. These data indicate that BBT is metabolized to at least two reactive compounds capable of covalent modification of protein and/or a single reactive product is formed which contains both the benzo ring (of benzotriazole) and the benzyl carbon atom (of the N-benzyl group); that P450 apoprotein modification may be an important mechanism of inactivation of pulmonary and hepatic P450 by BBT; and that hepatic microsomes from betaNF-induced guinea pigs generate more metabolites that do not act as mechanism-based P450 inhibitors from BBT than do those from PB-induced animals.
Subject(s)
Cytochrome P-450 Enzyme Inhibitors , Lung/enzymology , Microsomes, Liver/metabolism , Microsomes/metabolism , Triazoles/pharmacokinetics , Animals , Biotransformation , Carbon Radioisotopes , Enzyme Activation/drug effects , Gas Chromatography-Mass Spectrometry , Guinea Pigs , Male , Microsomes/enzymology , Microsomes, Liver/enzymology , Protein BindingABSTRACT
The kinetics of inactivation of cytochrome P450 2B1, the major phenobarbital inducible rat hepatic P450, by N-benzyl-1-aminobenzotriazole (BBT) were characterized. Purified, reconstituted P450 2B1 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylase activity was inhibited by BBT in a mechanism-based manner. The loss of O-deethylase activity followed pseudo-first-order kinetics and was NADPH and BBT dependent. After a 5 min incubation, greater than 90% of the 2B1 activity was lost, whereas more than 70% of the ability of the reduced enzyme to bind CO was maintained. Inclusion of 10 mM glutathione in the inactivation reaction lowered the rate of inactivation (k(inactivation)) and increased the partition ratio without significantly affecting the inactivator concentration required for half-maximal inactivation (K(I)). The maximal rate constant for inactivation at 23 degrees C was 0.24 min(-1) without and 0.15 min(-1) with glutathione. The apparent K(I) was 2 microM in both cases. The extrapolated partition ratios were 4 and 9 without and with 10 mM glutathione, respectively. Consistent with mechanism-based inactivation, the loss of 7-EFC O-deethylase activity was irreversible, was not due to product inhibition, was saturable, and could be slowed by including increasing concentrations of competing substrate. However, the inactivated P450 2B1 was still able to metabolize substrate if iodosobenzene was used as an alternate oxidant. Inactivation of 2B1 with either N-[14C]-7-benzyl-1-aminobenzotriazole (BBT) or N-benzyl-1-amino-[14C]-2,3-benzotriazole resulted in the incorporation of covalent radiolabel into the apoprotein. The stoichiometry of labeled metabolite adduct to protein was approximately 0.4:1 in both cases. Identification of metabolites revealed the formation of 1-aminobenzotriazole, benzotriazole, benzaldehyde, and a new metabolite (27) during catalysis of BBT by P450 2B1. Together, these data suggest that P450 2B1 could be inactivated and labeled by more than one metabolite.
Subject(s)
Cytochrome P-450 CYP2B1/antagonists & inhibitors , Triazoles/metabolism , Triazoles/toxicity , Animals , Apoproteins/metabolism , Coumarins/metabolism , Cytochrome P-450 CYP2B1/metabolism , Iodobenzenes/metabolism , Kinetics , Liver/drug effects , Liver/enzymology , Male , Protein Binding , Rats , Rats, Inbred Strains , Substrate SpecificityABSTRACT
OBJECTIVE: To investigate the single dose-response effects of grapefruit juice on terfenadine disposition and electrocardiographic measurements. METHODS: Twelve healthy males received 250 ml water or regular- or double-strength grapefruit juice with 60 mg terfenadine in a randomized crossover trial. Plasma concentrations of the cardiotoxic agent terfenadine and the active antihistaminic metabolite terfenadine carboxylate were determined over 8 hours. The QTc interval was monitored. RESULTS: Terfenadine concentrations were measurable (> 1 ng/ml) in 27 (20%; p < 0.001) and 39 (30%; p < 0.001) samples from individuals treated with regular- and double-strength grapefruit juice, respectively, compared to only four (3%) samples with water. Terfenadine plasma peak drug concentration (Cmax) was also higher. Terfenadine carboxylate area under the plasma drug concentration-time curve (AUC), Cmax, and time to reach Cmax (tmax) were increased by both strengths of juice. However, terfenadine carboxylate apparent elimination half-life (t1/2) was not altered. The magnitude of the interaction of terfenadine carboxylate AUC and Cmax ranged severalfold and correlated among individuals for regular-strength (r2 = 0.87; p < 0.0001) and double-strength (r2 = 0.78; p < 0.0001) grapefruit juice. No differences in the pharmacokinetics of terfenadine and terfenadine carboxylate were observed between the two strengths of grapefruit juice. QTc interval was not altered. CONCLUSIONS: A normal amount of regular-strength grapefruit juice produced maximum single-dose effects on terfenadine and carboxylic acid metabolite pharmacokinetics. The mechanism likely involved reduced presystemic drug elimination by inhibition of more than one metabolic pathway. The extent of the interaction was not sufficient to produce electrocardiographic changes. However, the pharmacokinetic effects were highly variable among individuals. This study further enhances the awareness of the potential for a serious interaction between grapefruit juice and terfenadine.
Subject(s)
Anti-Allergic Agents/pharmacokinetics , Anti-Asthmatic Agents/pharmacokinetics , Citrus/adverse effects , Food-Drug Interactions , Terfenadine/pharmacokinetics , Adult , Area Under Curve , Beverages , Electrocardiography/drug effects , Humans , Male , Oxidation-Reduction , Reference ValuesABSTRACT
Cytochrome P450 (P450) peroxygenase reactions are catalyzed by the ferric form of the enzyme and utilize hydroperoxides as oxidant donors. These reactions involve heterolytic cleavage of the hydroperoxide O-O bond and oxidation of the heme iron of P450 to a ferryl-oxyl complex, with subsequent transfer of the activated oxygen to a substrate. In the present study, we have demonstrated that arachidonic acid (AA) can serve as a substrate for P450 peroxygenase activity in the presence of cumene hydroperoxide (CuOOH) as a cosubstrate. AA is transformed into the same primary metabolite classes in both NADPH- and CuOOH-dependent oxidations. However, differences in the efficiency of the formation of the primary metabolite classes by CuOOH suggest that the disposition of AA in the presence of hydroperoxides is highly P450 isozyme dependent. Of particular note was the remarkable efficiency of AA epoxidation observed in the CuOOH-dependent reaction in guinea pig liver, whereas the formation of hydroxylated derivatives of AA at the thermodynamically less reactive positions C16 through C20 was more efficiently catalyzed via NADPH-dependent oxidation. Peroxygenase metabolism of AA and/or lipid hydroperoxide transformation reactions may be important functions for endothelial cells that contain P450 but are deficient in NADPH-P450 reductase, and they may also be involved in the cellular response to oxidative stress during NADPH depletion.
Subject(s)
Arachidonic Acid/metabolism , Animals , Benzene Derivatives/metabolism , Chromatography, High Pressure Liquid , Enzyme Induction/drug effects , Ferric Compounds , Guinea Pigs , Hydroxyeicosatetraenoic Acids/metabolism , Isoenzymes/metabolism , Male , Microsomes, Liver/metabolism , Mixed Function Oxygenases/metabolism , NADP/metabolism , Phenobarbital/pharmacology , beta-Naphthoflavone/pharmacologyABSTRACT
The time dependence for mechanism-based inactivation of cytochrome P450 (P450)-dependent 7-pentoxyresorufin O-depentylation (PROD), 7-ethoxyresorufin O-deethylation (EROD), and 7-methoxyresorufin O-demethylation (MROD) activities by N-benzyl-1-aminobenzotriazole (BBT) and N-alpha-methylbenzyl-1-aminobenzotriazole (alpha MB) was investigated in hepatic and pulmonary microsomes from phenobarbital-treated guinea pigs. In the presence of NADPH, both compounds inhibited P450-dependent catalytic activity in a time- and concentration-dependent manner. Inactivation of hepatic PROD activity was more rapid (t1/2 = 13.2 vs. 155 min) for 0.1 microM alpha MB when compared with equimolar BBT. On the other hand, hepatic EROD inactivation was more rapid (t1/2 = 8.1 vs. 11 min) with 0.1 microM BBT, compared with equimolar alpha MB. Inactivation of pulmonary PROD activity was the most rapid and potent, with an apparent half-life for inactivation of t1/2 = 0.94 and 32.2 min for 0.025 microM alpha MB and BBT, respectively. Incubation of hepatic microsomes for 45 min in the presence of NADPH and 10 microM BBT or alpha MB resulted in > 90% inhibition of PROD, EROD, and MROD activities. After washing by repeated sedimentation and resuspension, inhibition of PROD (78%; 93% for BBT and alpha MB, respectively), EROD (80% and 50%), and MROD (15% and 3%) activities was reversed to varying degrees. We conclude that BBT and alpha MB are rapidly metabolized to products that inhibit individual P450 isozymes by both mechanism-based (P4502B and P4501A1) and reversible (P4501A2) mechanisms. Of the two inhibitors, alpha MB is relatively more potent and selective for guinea pig lung P4502B isozyme(s).
Subject(s)
Cytochrome P-450 Enzyme Inhibitors , Isoenzymes/antagonists & inhibitors , Liver/drug effects , Lung/drug effects , Triazoles/pharmacology , Animals , Cytochrome P-450 CYP1A1/antagonists & inhibitors , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP1A2/metabolism , Cytochrome P-450 CYP1A2 Inhibitors , Cytochrome P-450 Enzyme System/metabolism , Guinea Pigs , In Vitro Techniques , Isoenzymes/metabolism , Kinetics , Liver/enzymology , Lung/enzymology , Male , NADP/metabolism , Oxazines/metabolismABSTRACT
OBJECTIVE: To investigate a potentially marked effect by erythromycin on felodipine pharmacokinetics, to characterize the mechanism, and to compare the interaction with that between grapefruit juice and felodipine. METHODS: Felodipine, 10 mg extended release, was administered with 250 ml water, 250 mg erythromycin, or 250 ml grapefruit juice in a randomized crossover study of 12 healthy men. Erythromycin base, 250 mg four times a day, was started the day before and continued on that study day. Pharmacokinetic values of felodipine, the primary metabolite dehydrofelodipine, and the major secondary derivative M3 metabolite were studied. RESULTS: Compared with water, erythromycin produced severalfold higher felodipine area under the plasma drug concentration-time profile (AUC), plasma peak drug concentration (Cmax), and apparent elimination half-life (t1/2); however, the effect was variable among individuals. Erythromycin augmented dehydrofelodipine AUC, Cmax, and t1/2 but decreased dehydrofelodipine/felodipine ratios. The AUC of the M3 metabolite and the M3 metabolite/dehydrofelodipine ratios were reduced. These findings support inhibition of both metabolic pathways likely mediated by CYP3A4. Grapefruit juice produced similar mean effects but did not prolong felodipine or dehydrofelodipine t1/2. Individually, felodipine AUC with erythromycin was greater than or similar to that with grapefruit juice. Relative felodipine AUC (erythromycin compared with grapefruit juice) correlated with relative felodipine Cmax but not with relative felodipine t1/2, suggesting felodipine AUC differed between these treatments, mainly from factors affecting presystemic drug elimination. CONCLUSIONS: Erythromycin produced an important pharmacokinetic interaction with felodipine by inhibition of drug metabolism. Although erythromycin and grapefruit juice shared a common mechanism, erythromycin likely reduced felodipine biotransformation at the gut wall and liver, whereas single-dose grapefruit juice had an effect mainly at the gut wall.
Subject(s)
Anti-Bacterial Agents/pharmacology , Beverages , Calcium Channel Blockers/pharmacokinetics , Citrus , Erythromycin/pharmacology , Felodipine/pharmacokinetics , Adolescent , Adult , Cross-Over Studies , Cytochrome P-450 Enzyme System/physiology , Drug Interactions , Humans , MaleSubject(s)
Eicosanoids/metabolism , Myocardial Ischemia/etiology , Myocardial Reperfusion Injury/etiology , Animals , Arachidonic Acid/metabolism , Cytochrome P-450 Enzyme System/metabolism , Epoxy Compounds/metabolism , Humans , Ischemic Preconditioning, Myocardial , Leukotrienes/metabolism , Lipoxygenase/metabolism , Myocardial Ischemia/metabolism , Myocardial Reperfusion Injury/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Prostaglandins/metabolismABSTRACT
1. Felodipine 10 mg extended release was administered with 250 ml regular-strength grapefruit juice or water in a randomized crossover manner followed by a second grapefruit juice treatment in 12 healthy men. The pharmacokinetics of felodipine and primary oxidative metabolite, dehydrofelodipine, were evaluated. 2. Initial grapefruit juice treatment increased felodipine AUC (mean +/- s.d.; 56.6 +/- 21.9 vs 28.1 +/- 11.5 ng ml-1 h; P < 0.001) and Cmax (8.1 +/- 2.5 vs 3.3 +/- 1.2 ng ml-1; P < 0.001) compared with water. Felodipine tmax (median; 2.8 vs 3.0 h) and t1/2 (7.3 +/- 3.7 vs 6.9 +/- 3.6 h) were not altered. 3. Readministration of felodipine with grapefruit juice produced mean felodipine AUC (61.5 +/- 32.2 ng ml-1 h) and Cmax (8.4 +/- 4.8 ng ml-1) which were similar to the initial grapefruit juice treatment 1-3 weeks previously. Felodipine AUC (r = 0.73, P < 0.01) and Cmax (r = 0.69, P < 0.02) correlated between grapefruit juice treatments among individuals. 4. The % increase in felodipine AUC with the initial grapefruit juice treatment compared with water correlated with the % increase in felodipine Cmax among individuals (r = 0.80, P < 0.01). Dehydrofelodipine AUC (74.7 +/- 28.7 vs 48.5 +/- 16.3 ng ml-1 h; P < 0.01) and Cmax (12.1 +/- 2.9 vs 7.9 +/- 2.6 ng ml-1; P < 0.01) were augmented with grapefruit juice compared with water.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Calcium Channel Blockers/pharmacokinetics , Citrus , Felodipine/analogs & derivatives , Felodipine/pharmacokinetics , Adult , Analysis of Variance , Beverages , Citrus/chemistry , Cross-Over Studies , Delayed-Action Preparations , Felodipine/administration & dosage , Felodipine/metabolism , Food-Drug Interactions , Humans , Linear Models , Male , Reproducibility of Results , White PeopleABSTRACT
The kinetics of mechanism-based inactivation of phenobarbital-inducible rabbit hepatic cytochromes P450 2B4 and 2B5 by N-benzyl-(BBT) and N-alpha-methylbenzyl (alpha MB) 1-aminobenzotriazole were investigated using reconstituted P450 2B4, a stable heterologous expression system, and hepatic microsomes. Low micromolar concentrations of the 1-aminobenzotriazole derivatives caused reversible inhibition as well as rapid inactivation of reconstituted P450 2B4 and recombinant P450 2B4 and 2B5. In contrast, even at a 1000-fold higher concentration, aminobenzotriazole inactivated the expressed P450 2B enzymes less rapidly. Preincubation of phenobarbital-induced hepatic microsomes with BBT and alpha MB resulted in concentration-dependent decreases in marker activities of P450 2B4 and 2B5, benzyloxyresorufin O-debenzylase and androstenedione 15 alpha-hydroxylase, respectively. BBT caused the inactivation of P450 2B4 and 2B5 in hepatic microsomes with apparent Kl values of 1.9 and 2.4 microM and maximal rate constants of 0.29 and 0.18 min-1, respectively. alpha MB inactivated both P450 2B enzymes with similar Kl values (approximately 7 microM) and maximal rate constants only slightly higher for 2B4 compared with 2B5 (0.68 vs. 0.55 min-1). Similar P450 2B selectivity of BBT and alpha MB in both hepatic microsomes and the stable expression system further validates this new expression system and the use of the selective markers identified for 2B4 and 2B5 in hepatic microsomes. The results also provide a mechanistic basis for the high potency of the N-aralkylated 1-aminobenzotriazole derivatives in vivo and suggest that treatments that inactivate 2B4 will also lead to 2B5 inactivation.